Field assessment of the operating procedures of a semi-quantitative G6PD Biosensor to improve repeatability of routine testing

In remote communities, diagnosis of G6PD deficiency is challenging. We assessed the impact of modified test procedures and delayed testing for the point-of-care diagnostic STANDARD G6PD (SDBiosensor, RoK), and evaluated recommended cut-offs. We tested capillary blood from fingerpricks (Standard Method) and a microtainer (BD, USA; Method 1), venous blood from a vacutainer (BD, USA; Method 2), varied sample application methods (Methods 3), and used micropipettes rather than the test’s single-use pipette (Method 4). Repeatability was assessed by comparing median differences between paired measurements. All methods were tested 20 times under laboratory conditions on three volunteers. The Standard Method and the method with best repeatability were tested in Indonesia and Nepal. In Indonesia 60 participants were tested in duplicate by both methods, in Nepal 120 participants were tested in duplicate by either method. The adjusted male median (AMM) of the Biosensor Standard Method readings was defined as 100% activity. In Indonesia, the difference between paired readings of the Standard and modified methods was compared to assess the impact of delayed testing. In the pilot study repeatability didn’t differ significantly (p = 0.381); Method 3 showed lowest variability. One Nepalese participant had <30% activity, one Indonesian and 10 Nepalese participants had intermediate activity (≥30% to <70% activity). Repeatability didn’t differ significantly in Indonesia (Standard: 0.2U/gHb [IQR: 0.1–0.4]; Method 3: 0.3U/gHb [IQR: 0.1–0.5]; p = 0.425) or Nepal (Standard: 0.4U/gHb [IQR: 0.2–0.6]; Method 3: 0.3U/gHb [IQR: 0.1–0.6]; p = 0.330). Median G6PD measurements by Method 3 were 0.4U/gHb (IQR: -0.2 to 0.7, p = 0.005) higher after a 5-hour delay compared to the Standard Method. The definition of 100% activity by the Standard Method matched the manufacturer-recommended cut-off for 70% activity. We couldn’t improve repeatability. Delays of up to 5 hours didn’t result in a clinically relevant difference in measured G6PD activity. The manufacturer’s recommended cut-off for intermediate deficiency is conservative

The durability of long-lasting insecticidal nets distributed to the households between 2009 and 2013 in Nepal

Background: Understanding and improving the durability of long-lasting insecticidal nets (LLINs) in the field are critical for planning future implementation strategies including behavioral change for care and maintenance. LLIN distribution at high coverage is considered to be one of the adjunctive transmission reduction strategies in Nepal’s Malaria Strategic Plan 2014–2025. The main objective of this study was to assess the durability through assessment of community usage, physical integrity, residual bio-efficacy, and chemical retention in LLINs: Interceptor®, Yorkool®, and PermaNet ®2.0 which were used in Nepal during 2009 through 2013. Methods: Assessments were conducted on random samples (n = 440) of LLINs from the eleven districts representing four ecological zones: Terai plain region (Kailali and Kanchanpur districts), outer Terai fluvial ecosystem (Surkhet, Dang, and Rupandhei districts), inner Terai forest ecosystem (Mahhothari, Dhanusa, and Illam districts), and Hills and river valley (Kavrepalanchock and Sindhupalchok districts). For each LLIN, fabric integrity in terms of proportionate hole index (pHI) and residual bio-efficacy were assessed. However, for chemical retention, a representative sample of 44 nets (15 Yorkool®, 10 Permanet®2.0, and 19 Interceptor®) was evaluated. Data were analyzed using descriptive statistics stratified by LLINs brand, districts, and duration of exposure. Results: On average, duration of use of LLINs was shortest for the Yorkool® samples, followed by PermaNet® 2.0 and Interceptor® with median ages of 8.9 (IQR = 0.4), 23.8 (IQR = 3.2), and 50.1 (IQR = 3.2) months, respectively. Over 80% of field distributed Yorkool® and PermaNet® 2.0 nets were in good condition (pHI< 25) compared to Interceptor® (66%). Bio-efficacy analysis showed that average mortality rates of Interceptor and Yorkool were below World Health Organization (WHO) optimal effectiveness of ≥ 80% compared to 2-year-old PermaNet 2.0 which attained 80%. Chemical retention analysis was consistent with bio-efficacy results. Conclusion: This study shows that distribution of LLINs is effective for malaria control; however, serviceable life of LLINs should be considered in terms of waning residual bio-efficacy that warrants replacement. As an adjunctive malaria control tool, National Malaria Control Program of Nepal can benefit by renewing the distribution of LLINs in an appropriate time frame in addition to utilizing durable and effective LLINs

Co-Circulation of Dengue Virus Serotypes 1, 2, and 3 during the 2022 Dengue Outbreak in Nepal: A Cross-Sectional Study

The largest dengue outbreak in the history of Nepal occurred in 2022, with a significant number of casualties. It affected all 77 districts, with the nation’s capital, Kathmandu (altitude 1300 m), being the hardest hit. However, the molecular epidemiology of this outbreak, including the dengue virus (DENV) serotype(s) responsible for this epidemic, remain unknown. Here, we report the epidemic trends, clinico-laboratory features, and virus serotypes and their viral load profiles that are associated with this outbreak in Nepal. Dengue-suspected febrile patients were investigated by routine laboratory, serological, and molecular tools, including a real-time quantitative polymerase chain reaction (qRT-PCR). Of the 538 dengue-suspected patients enrolled, 401 (74.5%) were diagnosed with dengue. Among these dengue cases, 129 (32.2%) patients who required hospital admission had significant associations with myalgia, rash, diarrhea, retro-orbital pain, bleeding, and abdominal pain. DENV-1, -2, and -3 were identified during the 2022 epidemic, with a predominance of DENV-1 (57.1%) and DENV-3 (32.1%), exhibiting a new serotype addition. We found that multiple serotypes circulated in 2022, with a higher frequency of hospitalizations, more severe dengue, and more deaths than in the past. Therefore, precise mapping of dengue and other related infections through integrated disease surveillance, evaluation of the dynamics of population-level immunity and virus evolution should be the urgent plans of action for evidence-based policy-making for dengue control and prevention in the country

Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley

This cross-sectional study was designed to identify information on the frequency, antimicrobial resistance and species diversity of methicillin-resistant coagulase negative staphylococci (MRCoNS) among pet dogs and humans within households. Fifty five nasal swabs each from dogs and their owners were collected. MRCoNS were identified based on gram staining, culture on mannitol salt agar, biochemical tests, and mecA gene amplification. The antibiotic susceptibility of the isolates was assessed by a disc diffusion test. Uniplex and multiplex polymerase chain reaction (PCR) were employed for the species identification of MRCoNS and SCCmec typing, respectively. Species were further confirmed by MALDI-TOF-MS. The prevalence of MRCoNS was 29% in dog owners and 23.6% in dogs. Four different species of MRCoNS, Staphylococci saprophyticus (48.3%), S. haemolyticus (24.1%), S. warneri (17.2%), and S. epidermidis (10.3%), were detected. Two isolates each from dog owners and dogs showed a constitutive resistance to macrolide-lincosamide-streptogramin B (cMLSB) resistance, eight isolates each from dogs and their owners showed a macrolide-streptogramin B (MSB) resistance, and only two isolates from dog owners revealed an inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB) resistance. SCCmec types were SCCmec type IV (55.2%), SCCmec type V (24.1%), SCCmec III (10.3%), SCCmec II (3.4%); two isolates were non-typable. MRCoNS are prevalent and genetically diverse in companion animals and humans. Different species of MRCoNS were found in dogs and their owners

Methicillin-Resistant Coagulase Negative Staphylococci and Their Antibiotic Susceptibility Pattern from Healthy Dogs and Their Owners from Kathmandu Valley

This cross-sectional study was designed to identify information on the frequency, antimicrobial resistance and species diversity of methicillin-resistant coagulase negative staphylococci (MRCoNS) among pet dogs and humans within households. Fifty five nasal swabs each from dogs and their owners were collected. MRCoNS were identified based on gram staining, culture on mannitol salt agar, biochemical tests, and mecA gene amplification. The antibiotic susceptibility of the isolates was assessed by a disc diffusion test. Uniplex and multiplex polymerase chain reaction (PCR) were employed for the species identification of MRCoNS and SCCmec typing, respectively. Species were further confirmed by MALDI-TOF-MS. The prevalence of MRCoNS was 29% in dog owners and 23.6% in dogs. Four different species of MRCoNS, Staphylococci saprophyticus (48.3%), S. haemolyticus (24.1%), S. warneri (17.2%), and S. epidermidis (10.3%), were detected. Two isolates each from dog owners and dogs showed a constitutive resistance to macrolide-lincosamide-streptogramin B (cMLSB) resistance, eight isolates each from dogs and their owners showed a macrolide-streptogramin B (MSB) resistance, and only two isolates from dog owners revealed an inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB) resistance. SCCmec types were SCCmec type IV (55.2%), SCCmec type V (24.1%), SCCmec III (10.3%), SCCmec II (3.4%); two isolates were non-typable. MRCoNS are prevalent and genetically diverse in companion animals and humans. Different species of MRCoNS were found in dogs and their owners

Multidrug-resistant bacteria with ESBL genes: a growing threat among people living with HIV/AIDS in Nepal

BACKGROUND: Bacterial opportunistic infections are common in people living with HIV/AIDS (PLHA). Besides HIV-TB co-infection, lower respiratory tract infections (LRTIs) due to multidrug-resistant (MDR) bacteria cause significant morbidity and mortality among PLHA. This study identified bacterial co-infection of the lower respiratory tract and detected plasmid-mediated blaTEM and blaCTX-M genes among Extended-Spectrum β-Lactamase (ESBL) producing isolates from sputum samples in PLHA. METHODS: A total of 263 PLHA with LRTIs were enrolled in this study, out of which, 50 were smokers, 70 had previous pulmonary tuberculosis, and 21 had CD4 count &lt; 200 cells/µl. Sputum samples collected from PLHA were processed with standard microbiological methods to identify the possible bacterial pathogens. The identified bacterial isolates were assessed for antibiotic susceptibility pattern using modified Kirby Bauer disk diffusion method following Clinical Laboratory Standard Institute (CLSI) guidelines. In addition, plasmid DNA was extracted from MDR and ESBL producers for screening of ESBL genes; blaCTX-M and blaTEM by conventional PCR method using specific primers. RESULTS: Of 263 sputum samples, 67 (25.48%) showed bacterial growth. Among different bacterial pathogens, Klebsiella pneumoniae, (17; 25.37%) was the most predominant, followed by Haemophillus influenzae, (14; 20.90%) and Escherichia coli, (12; 17.91%). A higher infection rate (4/8; 50%) was observed among people aged 61–70 years, whereas no infection was observed below 20 years. About 30.0% (15/50) of smokers, 32.86% (23/70) cases with previous pulmonary tuberculosis, and 52.38% (11/21) with CD4 count &lt; 200 cells/µl had bacterial LRTIs. Among 53 bacterial isolates excluding H. influenzae, 28 isolates were MDR and 23 were ESBL producers. All ESBL producers were sensitive to colistin and polymyxin B. Among ESBL producers, 47.83% (11/23) possessed blaCTX-M, 8.6% (2/23) were positive for blaTEM gene, and 43.48% (10/23) possessed both ESBL genes. CONCLUSION: The increasing rate of MDR bacterial infections, mainly ESBL producers of LRTIs causes difficulty in disease management, leading to high morbidity and mortality of PLHA. Hence, it is crucial to know the antibiogram pattern of the isolates to recommend effective antimicrobial therapy to treat LRTIs in PLHA

Evaluation of Xpert MTB/RIF Assay, MTB Culture and Line Probe Assay for the Detection of MDR Tuberculosis in AFB Smear Negative Specimens

The global burden of tuberculosis (TB), particularly with multidrug resistance (MDR), is escalating and has become a major health challenge. It is well known that acid-fast bacilli (AFB) smear-negative TB patients are the major source of spreading TB to healthy individuals when left untreated. Early diagnosis of TB and rapid detection of drug resistance are important for the proper management of drug-resistant TB (DR-TB). Therefore, a laboratory based cross-sectional study was conducted from July to December 2019 at the National Tuberculosis Centre, Thimi, Nepal, with the objective of evaluating the diagnostic performance of Xpert MTB/RIF assay, Mycobacterium tuberculosis (MTB) culture and line probe assay (LPA) for the detection of MDR-TB in AFB smear-negative sputum samples. We evaluated a total of 222 AFB smear-negative sputum specimens, of which 21.6% (n = 48) showed MTB positive with Xpert MTB/RIF assay and, while culturing on Lowenstein–Jensen (LJ) media, 21.2% (n = 47) were MTB culture positive. The sensitivity, specificity, PPV and NPV at 95% confidence interval of Xpert MTB/RIF assay on diagnosing M. tuberculosis from smear-negative specimens were 73% (57–84), 92% (87–96), 71% (59–81) and 93% (89–95), respectively. In addition, the sensitivity of Xpert MTB/RIF assay and LPA in detecting rifampicin resistance was 75% (42–94, 95% CI) and 91.67% (62–99, 95% CI), respectively. The current study also assessed a significant association between the occurrence of pulmonary tuberculosis with different age group, TB history and alcohol consumption. These findings indicate that Xpert MTB/RIF assay and LPA are appropriate methods for early detection and accurate diagnosis of TB and RIF mono-resistant cases

Genotypically Confirmed Vancomycin-Resistant With Gene Among Clinical Isolates in Kathmandu

Purpose: Methicillin-resistant Staphylococcus aureus , a common bacterial pathogen causes various infections. The acquisition of various antimicrobial-resistant genes in S. aureus has led to the transformation of this bacterium into a superbug. Vancomycin resistance among MRSA isolates is an emerging threat in empirical therapy of various infections. The study was hence aimed to find out the susceptibility status of S. aureus isolates toward vancomycin and detect mecA, vanA , and vanB genes among the isolates. Methods: A total of 1245 clinical samples from the participants attending a tertiary care hospital in Kathmandu were processed. S. aureus isolated from the samples were subjected to antibiotic susceptibility patterns using the modified Kirby-Bauer disk diffusion method. Agar dilution method was used to determine the minimum inhibitory concentration of vancomycin. The antibiotic-resistant genes such as mecA, vanA , and vanB among S. aureus isolates were screened by a conventional polymerase chain reaction. Results: Of 1245 samples, 80 S. aureus were identified. Out of which, 47.5% (38/80) were phenotypically confirmed MRSA isolates. mec A gene was detected in 84.2% (32/38) of MRSA isolates. 10.5% (4/38) were confirmed as vancomycin-intermediate S. aureus (VISA) by MIC determination. None of the isolates was positive for the vanA gene; however, 2 isolates were found to possess the vanB gene. The 2 isolates have vancomycin MIC breakpoints of 4 to 8 μg/mL. Conclusion: There might be a spreading of vancomycin resistance among S. aureus , creating serious public health problems. Therefore, measures to limit vancomycin resistance should be considered in healthcare facilities as immediately as possible